This invention relates to a method of obtaining pinitol from carob extracts using chromatographic techniques on ion-exchange resins.
Pinitol is a cyclitol corresponding to the methylated form of D-chiro-inositol and more concretely it is 3-O-methyl-1,2,4 cis-3,5,6 transhexahydroxycyclohexanol.
The name pinitol derives from xe2x80x9cpinexe2x80x9d, as it had been isolated from this tree. (Ballou C. E., Anderson A. B., J. Am. Chem. Soc. 75, 648-670 (1953)). It also occurs widely in other plants such as: soya (Phillips D. V., Smith A. E., J. Agric. Food Chem. 30, 456-466 (1982)), in leaves of Bougainvillea spectabilis (Narayana C. R., Joshi D. D., Mujumdar A. M., Dhekne V. V., Current Science 56, 130-141 (1987)), in leaves of Gliricidia sepium (Calle J., Rivera A., Joseph-Nathan P., Planta Med. 53, (3), 303 (1987)), etc.
Pinitol can also be obtained by chemical synthesis, but until now the process has been very expensive.
Pinitol is a normal component of the human diet. It is present in soya at about 1% of dry weight (Ostlund et al., U.S. Pat. No. 5,550,166). In some Asian countries, where consumption of soya is very widespread, it is calculated that the consumption of pinitol, via soya, is greater than 5 mg/kg/day.
In PCT WO 00/71111 A1 of Humanectics Corporation, it is claimed that pinitol enhances the function of muscle tissue, increases the formation of glycogen in muscle and stimulates the transport of glucose within muscle tissue.
The use of pinitol for the treatment of disorders associated with resistance to insulin is claimed in PCT WO 96/29063 of Washington University. Therefore, pinitol can be used for treatments connected with diabetes mellitus and its chronic complications; obesity, hyperlipidemias, dyslipidemias, atherosclerosis, hypertension, cardiovascular disorders, AIDS, cancer, malnutrition, stress, lupus and other autoimmune disorders, endocrin disorders and complications arising from athletic activity or from inactivity.
Pinitol is used at a dose from 0.1 mg to 1.0 g per day per kg of weight. It can be administered orally, parenterally or intravenously.
The aforementioned document PCT WO 96/29063 also describes a method of obtaining pinitol from soluble fractions of soya, in which said fractions are first deproteinized and treated with active carbon before undergoing column chromatography.
According to this known method, after deproteinization and treatment with active carbon, the material is deionized by passing it over ion-exchange resins and then the sugars are separated by means of anionic resins. The resulting fraction, rich in cyclitols, is employed for pharmacological tests.
On the other hand, U.S. Pat. No. 5,482,631 describes a method of separating inositols and sugars from an aqueous phase of sugar cane molasses, beet, almonds and soya, which comprises passing said aqueous phase over a strong anionic exchange resin of the hydroxide form. The process is preferably carried out in a simulated moving-bed chromatographic system, details of which will be given later.
As is clear from the state of the art noted above, production of pinitol basically employs ion-exchange techniques using strong anionic exchange resins of the OH form, and starting from materials such as soya, sugar cane molasses, beet and almonds.
In addition to WO 96/29063 and U.S. Pat. No. 5,482,631, the use of strong anionic resins of the OH form for the separation of carbohydrates is mentioned in Roseman et al., Arch. Biochem. Biophys. 36, 232, (1952); and Phillips et al., J. Agric. Food Chem. 30, 456-458 (1982).
The use of resins of this type has the advantage of good separation of the cyclitols from the carbohydrates. On the other hand, it has serious drawbacks. Firstly, there is decomposition of the sugars in an alkaline medium and, secondly, the adsorption often has a high degree of irreversibility, all of which gives rise to difficulties for the regeneration of these resins and hence a very short useful life and very high costs.
Accordingly, it is more interesting to use strong cationic resins (Na, K, Li, Ca, etc.), which are more stable and for which the adsorption effects are clearly reversible.
Thus, patent ES 2060544 describes a method for obtaining a syrup consisting of the natural sugars of the carob, which comprises extracting the sugars from carob pulp and submitting the juice thus obtained to chromatographic separation to separate the sugars from the non-sugars.
Before commenting on the method of patent ES 2060544, it should be pointed out that carob, used as the starting material in said method, is the fruit of the carob tree (Ceratonia siliqua), a slow-growing tree of medium size, evergreen, originating from the Mediterranean area. It belongs to the leguminosae and is the only member of the genus ceratonia. It grows in dry environments. Its pods are the carob beans. The carob tree is encountered abundantly throughout the Mediterranean basin. Spain is the largest producer of carob, with almost 50% of the total.
The use of carob as a human and/or animal food product has been known since antiquity. It has a long history of application as foodstuff and as a pharmaceutical product.
It has also found wide application as a cocoa substitute. Relative to cocoa, it has the advantage that it is not allergenic, and does not produce the effect of addiction of caffeine and theobromine. It contains less fat and more sugars.
The yellowish-white powder of the carob seed (GARROFIN) is used as an additive in the food industry (ice creams, jams, etc.).
Carobs are characterized as being rich in sugars. The average amount of saccharose, glucose and fructose is in the range 40-50% based on dry matter of the carob.
These comments concerning carob having been made, the method of patent ES 2060544 comprises a series of stages for extracting the sugars from the carob pulp, which include, in summarized form:
cleaning of the carob; cutting into pieces; classification; extraction with water; pressing of the husks; prefiltration; decalcification; fine filtration; evaporation and preliminary concentration; chromatographic separation of sugars and non-sugars; demineralization and decolorizing by resins; and evaporation and final concentration.
Concretely, in relation to the stage of chromatographic separation of sugars and non-sugars, patent ES 2060544 employs a strong cationic resin, based on weakly crosslinked polystyrene, whose active sulfonic groups are charged with a monovalent or divalent cation, thus arriving at a carob syrup with the following composition:
The syrup thus obtained consists of the natural sugars of the carob and is free of the adverse characteristics of color, odor and flavor of the natural extract of carob.
According to Saura Calixto F (An. Bromatol. 39 (1), 81-93 (1987)), analysis of carob pulp by HPLC and gas chromatography gives the result:
In view of these data, it would be desirable to have a method of obtaining pinitol from carob extracts.
In this connection, a method is known for the production of pinitol by fermentation of carob extracts. Thus, Baumgartner et al. (J. Agric. Food Chem., 34, 827-829 (1986)) ferment carob extracts with Saccharomyces bayanus at 30xc2x0 C. for seven days. Pinitol is isolated and identified from the fermented product. Analyses by TLC, gas chromatography and GC-MS made it possible to determine the composition of the fermented carob extract, which was:
It has now been demonstrated that, starting from the carob syrup obtained according to patent ES 2060544, it is possible to separate and recover pinitol by means of ion-exchange resins, both strong anionic resins and strong cationic resins.
Accordingly, the aim of the present invention is to provide a method of obtaining pinitol starting from carob extracts that have the composition:
in which the percentages are expressed in weight of dry matter, distinguished by the fact that, once the saccharose contained in the extract has been inverted to fructose and glucose, the resulting syrup is submitted to chromatographic separation of the pinitol by means of ion-exchange resins, strong anionic or strong cationic, to obtain a solution of pinitol in water, after which the pinitol is separated from said solution. Preferably, the pinitol is separated from said solution by concentration and addition of an organic solvent or by atomization and freeze-drying.